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. 2000 Mar 1;19(5):1079-86.
doi: 10.1093/emboj/19.5.1079.

Cap-dependent deadenylation of mRNA

E Dehlin  1 , M Wormington, C G Körner, E Wahle
Affiliations

Cap-dependent deadenylation of mRNA

E Dehlin et al. EMBO J. .

Abstract

Poly(A) tail removal is often the initial and rate-limiting step in mRNA decay and is also responsible for translational silencing of maternal mRNAs during oocyte maturation and early development. Here we report that deadenylation in HeLa cell extracts and by a purified mammalian poly(A)-specific exoribonuclease, PARN (previously designated deadenylating nuclease, DAN), is stimulated by the presence of an m(7)-guanosine cap on substrate RNAs. Known cap-binding proteins, such as eIF4E and the nuclear cap-binding complex, are not detectable in the enzyme preparation, and PARN itself binds to m(7)GTP-Sepharose and is eluted specifically with the cap analog m(7)GTP. Xenopus PARN is known to catalyze mRNA deadenylation during oocyte maturation. The enzyme is depleted from oocyte extract with m(7)GTP-Sepharose, can be photocross-linked to the m(7)GpppG cap and deadenylates m(7)GpppG-capped RNAs more efficiently than ApppG-capped RNAs both in vitro and in vivo. These data provide additional evidence that PARN is responsible for deadenylation during oocyte maturation and suggest that interactions between 5' cap and 3' poly(A) tail may integrate translational efficiency with mRNA stability.

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Figures

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Fig. 1. (A) Cap-dependent deadenylation in a HeLa cytoplasmic extract. Two reaction mixtures were assembled on ice with 36 fmol of either m7GpppG-capped or ApppG-capped, polyadenylated L3 pre-RNA in 70 μl of reaction buffer. The reaction was started by the addition of 1.7 μl of a HeLa cell cytoplasmic extract and incubation at 37°C. Aliquots were taken at the times indicated and RNAs analyzed on an 8% denaturing polyacrylamide gel. Substrate, decay products and completely deadenylated L3 pre-RNA are indicated by arrowheads. Sizes (in nucleotides) of the DNA marker are shown on the right. (B) Competition with free cap in HeLa cell cytoplasmic extract. The 20 μl reaction mixtures were assembled on ice with 10 fmol of m7G-capped and polyadenylated (A160) L3 pre-RNA, 0.5 μl of HeLa cell cytoplasmic extract and 0, 20, 100 or 330 μM m7GpppG or GpppG. The deadenylation reaction was carried out at 37°C for 80 min. Products were separated on an 8% denaturing polyacrylamide gel and analyzed on a PhosphorImager. Lane 1, substrate RNA lacking a poly(A) tail; lane 2, polyadenylated substrate RNA without incubation; lane 3, DNA size marker; the sizes of two fragments are indicated on the left. Deadenylation intermediates are indicated by arrowheads on the right.
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Fig. 1. (A) Cap-dependent deadenylation in a HeLa cytoplasmic extract. Two reaction mixtures were assembled on ice with 36 fmol of either m7GpppG-capped or ApppG-capped, polyadenylated L3 pre-RNA in 70 μl of reaction buffer. The reaction was started by the addition of 1.7 μl of a HeLa cell cytoplasmic extract and incubation at 37°C. Aliquots were taken at the times indicated and RNAs analyzed on an 8% denaturing polyacrylamide gel. Substrate, decay products and completely deadenylated L3 pre-RNA are indicated by arrowheads. Sizes (in nucleotides) of the DNA marker are shown on the right. (B) Competition with free cap in HeLa cell cytoplasmic extract. The 20 μl reaction mixtures were assembled on ice with 10 fmol of m7G-capped and polyadenylated (A160) L3 pre-RNA, 0.5 μl of HeLa cell cytoplasmic extract and 0, 20, 100 or 330 μM m7GpppG or GpppG. The deadenylation reaction was carried out at 37°C for 80 min. Products were separated on an 8% denaturing polyacrylamide gel and analyzed on a PhosphorImager. Lane 1, substrate RNA lacking a poly(A) tail; lane 2, polyadenylated substrate RNA without incubation; lane 3, DNA size marker; the sizes of two fragments are indicated on the left. Deadenylation intermediates are indicated by arrowheads on the right.
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Fig. 2. Purified PARN prefers correctly capped RNA. (A) The 50 μl reaction mixtures were assembled on ice containing 15 fmol of bovine PARN and 40 fmol of polyadenylated (A130) β-globin 3′-UTR RNA with an m7GpppG, GpppG, ApppG dinucleotide or pppG at the 5′ end of the transcript. The reactions were started by the addition of enzyme and incubation at 37°C. Then 7 μl aliquots were withdrawn at the time points indicated in the figure, and decay products were separated on a 6% denaturing polyacrylamide gel and analyzed on a PhosphorImager. Fully deadenylated β-globin RNA is indicated by an arrowhead, and the sizes of the DNA marker are shown at the side of the M lane. (B) Quantitation of the accumulation of fully deadenylated RNA in (A). The appearance of deadenylated substrate at each time point, as part of the total radioactivity in each lane, was quantitated on a PhosphorImager and plotted versus incubation time.
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Fig. 2. Purified PARN prefers correctly capped RNA. (A) The 50 μl reaction mixtures were assembled on ice containing 15 fmol of bovine PARN and 40 fmol of polyadenylated (A130) β-globin 3′-UTR RNA with an m7GpppG, GpppG, ApppG dinucleotide or pppG at the 5′ end of the transcript. The reactions were started by the addition of enzyme and incubation at 37°C. Then 7 μl aliquots were withdrawn at the time points indicated in the figure, and decay products were separated on a 6% denaturing polyacrylamide gel and analyzed on a PhosphorImager. Fully deadenylated β-globin RNA is indicated by an arrowhead, and the sizes of the DNA marker are shown at the side of the M lane. (B) Quantitation of the accumulation of fully deadenylated RNA in (A). The appearance of deadenylated substrate at each time point, as part of the total radioactivity in each lane, was quantitated on a PhosphorImager and plotted versus incubation time.
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Fig. 3. Inhibition of purified PARN by free m7GpppG. A reaction mixture was assembled on ice containing 95 fmol of m7G-capped and polyadenylated (A160) L3 pre-RNA and 60 fmol of bovine PARN in 160 μl of reaction buffer. The mix was divided into 20 μl aliquots and m7GpppG or GpppG was added at the concentrations indicated. The reactions were started by incubation at 37°C. After 90 min, they were stopped and products analyzed on a denaturing 8% polyacrylamide gel. The sizes of DNA markers are indicated. The position of fully deadenylated L3 pre-RNA (m7capL3pre) is indicated by an arrowhead. Note that the amount of PARN used was sufficient to degrade most of the body of the RNA; fully deadenylated RNA became visible upon partial inhibition by intermediate concentrations of free cap (see the text).
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Fig. 4. Cap-dependent deadenylation by Xenopus PARN. (A) In vivo deadenylation in mature oocytes. Gel-purified, radiolabeled G52 RNA containing 5′-terminal m7GpppG (lanes 1–3), ApppG (lanes 4–6) or GpppG (lanes 7–9) structures was injected into progesterone-matured Xenopus oocytes. RNA was isolated from oocytes 2, 4 or 6 h post-injection and analyzed by electrophoresis in 6% polyacrylamide–7 M urea gels. (B) In vitro deadenylation in Xenopus oocyte extracts. Oocyte extracts were incubated at 25°C for the indicated times with gel-purified, radiolabeled G52 RNA containing 5′-terminal m7GpppG (lanes 1 and 2), GpppG (lanes 3 and 4) or ApppG (lanes 5 and 6) structures.
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Fig. 5. Binding of bovine PARN to m7GTP–Sepharose. The experiment was carried out with a partially purified fraction of PARN as described in Materials and methods. Lane L (load), starting material; lane FT (flow-through), unbound proteins in the supernatant; lanes W1 and W2, washes with nucleotide-free buffer; lane GTP-W, GTP wash; lane m7GTP-W, m7GTP wash; lane B, beads; lane M, molecular weight markers. Proteins were detected by silver staining. Note that only 50% of the total sample was loaded in lanes L and FT, whereas the entire samples were loaded in the other lanes.
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Fig. 6. Binding of Xenopus PARN to m7GpppG cap. (A) Photochemical cross-linking of Xenopus p74 and p62 PARN isoforms to [32P]cap-labeled RNA. Total mature oocyte extract was incubated with 20 fmol of G52 RNA containing a radiolabeled m7GpppG cap in the presence of 0, 50 or 100 μM m7GpppG. After UV irradation and digestion with RNase A, samples were immuno- precipitated with anti-PARN 205-5 antibody and analyzed by 10% SDS–PAGE. (B) m7GTP–Sepharose depletion of Xenopus PARN. Total oocyte extract was incubated with m7GTP–Sepharose for 2 h at 4°C. Fractions corresponding to two oocyte equivalents of total (T), depleted supernatant (S) and bound (P) protein eluted with 0.5 mM m7GpppG were analyzed by immunoblot probed with anti-PARN 205-5 antibody (lanes 1–3). In addition, total (T) and m7GTP–Sepharose-depleted extract was analyzed for deadenylation activity by incubation for 1 h at 25°C with radiolabeled m7GpppG-capped G52 substrate RNA.
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